The human immune system is an elegant and complex system which protects the human body from foreign molecules and organisms; it also greatly limits the efficacy of otherwise life-saving organ transplants. The immune system is the organic system in the body which is badly damaged by acquired immunodeficiency syndrome (AIDS). The immune system may marshall armies of cells to ward off infection and/or induce responses which range from the inconvenient to the disabling.
The general sequence of the various immune responses, the cells and molecules involved, and certain of the immune response modulating factors are now generally understood, but much remains to be learned respecting specific cells, enhancing and suppressing factors, and the regulation of the immune response.
The immune response includes several molecules and cells which act, to greater or lesser degrees, to interdict immunogenic agents, typically allergens or pathogens, and/or to build a defense system to the particular immunogenic pathogen. Neutrophils, produced in the bone marrow, scavenge foreign molecules and organisms which carry antigenic determinants and the complement system may attach to the invading foreign species. Maerophages also engulf foreign molecules and cells, as well as debris from the body's own cells, and display the antigenic determinants of the invader thus signalling other cellular constituents of the immune system to respond.
T cells carry an array of receptors which recognize certain specific antigens, and the T cell system generally constitutes one of the key constituents of the overall immune system. There are three basic classes of T cells, referred to as Helper T cells, Suppressor T cells and Killer T cells. Helper T cells are activated to reproduce by a protein released by the macrophage, interleukin-1, II-1, and they produce a variety of interleukins which activate other T cells and B cells. Killer T cells attack and destroy certain immunogenic cells such as tissue from foreign sources, parasites, and immunogen containing cells such as virus-infected cells. Suppressor T cells modulate the immune response to meet the challenge from the foreign invader without an over-response.
B cells, stimulated by T cells, produce antibodies which couple very specifically to the antigenic determinants of the invading species. Antibodies coupled to the antigen inactivate the antigen bearing cell and/or makes it more vulnerable to ingestion or destruction by macrophages, neutrophils, and other elements of the immune system, and they activate the complement system to increase the attack upon the invading species. Both B cells and T cells contribute to a residual immunity to the particular antigen which may last for a few weeks or a life time.
The present invention contemplates the transfer of the critical functions of the immune response system described generally described above from a human, or one animal, to a mouse or a laboratory animal of a different species by the transfer and in vivo nurturing of cells of organs or fluids which express the immune function. The two main cell types involved in vertebrate immunity are phagocytes and lymphocytes. Phagocytic cells are sometimes referred to as the reticuloendothelial system, and more recently as the mononuclear phagocyte system, while lymphocytes, their precursors, derivatives and supportive cells are referred to as the lymphoid system. Cells from these systems may be used in the transfer of the immune functional system from the human, or other donor species, to a SCID mouse, SCID horse, or other laboratory animal, different from the donor species whose immune system is to be studied. The mononuclear phagocyte system, (MPS), includes cells involved in defense-related phagocytosis derived from bone marrow precursor cells characterized by involvement of immunoglobulins and serum complement. The organs in which most MPS cells are localized are the bone marrow, peripheral blood, connective tissue, liver, lungs, lymph nodes spleen and nervous system.
The lymphoid system comprises the tissues and organs in which lymphocyte precursors and their derivatives originate, mature, lodge and are moved. The principal organs of the lymphoid system of an adult human are, in addition to the lymphatic and blood vessels, the thymus, spleen, bone marrow, the lymphatic nodes, e.g. cervical, axillary, lumbar, iliac and inguinal nodes, and to a lesser extent the heart and stomach. The fetal lymphoid system includes the yolk sac, pharyngeal pouches, bone marrow, liver, spleen and thymus.
The present invention may involve the transfer of cells of any of or more than one of the above organs of the immune system.
Among the goals and features of the present invention is the evaluation of drugs, the development of therapeutic materials, the development of immunizing compositions, and the general study of methods for preventing, treating, enhancing or suppressing immune response to various foreign materials which are or may become pathogenic or undesirable in humans or other donor species whose immune system is to be studied. An important application of this principle is the development of vaccines and treatments for pathogenic organisms. The antigenic determinants of some pathogenic organisms remain constant from generation to generation and it is possible to induce immunity to diseases caused by such organisms; indeed, by vaccination many of the diseases which ravaged the world in past centuries have been essentially eliminated as a threat to the: earth's human population. Other organisms, such as influenza virus, however, mutate rapidly and/or are not single species but, rather, constitute a large number of species which differ immunologically. A vaccine to a single form of such species does not induce a lasting immunity to all forms of the species.
Successful development of vaccines and therapeutics for organisms and materials which trigger the immune response requires the ability to track the immune response upon challenge by such organisms and materials. It is, in most instances, possible to track the human immune response in several ways, more or less completely. The most complete tracking of the immune response would, theoretically, be possible in the human body, where the entire immune system resides. This is not generally convenient, however, even under the best of circumstances, where the invading species is not fatal, because it is generally impractical to use the human body as an immune system host for long term studies. It is, therefore, common practice to use in vitro cultures of human cells for studies of the human immune system. This approach, however, has limitationsl several features of the complex immune system do not function well in culture. Another approach is to study and/or the immune system of other species, mice, rats or guinea pigs typically.
It would be a great step forward in the study of the human immune system to have available a laboratory animal which could be husbanded in a controlled environment and which would provide an adequate in vivo host for the human immune system. One of the important features of this invention is the development of just such a laboratory animal, examples of which SCID animals, e.g. mice, in which the essential elements of the human immune system resides and thus produces human antibodies when challenged with particular immunogens.
One particular example where the availability of a functioning human immune system in mice, or another laboratory animal, would be a major advantage is in AIDS research. Currently, the most difficult challenge to immunologists has come with the spread of the Human Immunodeficiency Virus, HIV, (also referred to, generally in earlier reports, as lymphadenopathy-associated virus, LAV, and human-T-lymphotropic virus, HTLV, and acquired immune deficiency syndrome (AIDS) related virus, ARV) which is generally recognized as causing acquired immunodeficiency syndrome AIDS at least two HIV viruses, HIV-1 and HIV-2, have been identified as AIDS infective agents. These retroviruses, in which the genetic material is RNA rather than DNA, carry an enzyme which catalyzes transcription of vital RNA into DNA in the host. The DNA may then be integrated into the genome of an infected cell where it can then be inherited by daughter cells and form new virus particles. Through mechanisms not fully understood in humans, the HIV virus disrupts the normal immune response by depleting T helper/inducer cells, specifically T4 or CD4.sup.+ cells, which are responsible for the induction of most, if not all, of the functions of the human immune system. One of the first and ultimately most lethal events in the progress of AIDS is the suppression of much or all of the human immune system, leaving the body with little or no defense against the constant attack upon the human body by bacteria, viruses, plant and animal produced antigens, chemicals and foreign materials generally.
The study of the immunology of HIV is more challenging than the study of more traditional immunogenic materials for several reasons. All, or nearly all, of those in whom HIV induces the AIDS set of symptoms die within one to two years and are susceptible to most infectious organisms which are of little threat to those with a normally functioning immune system. Thus, the human body which is, at best, a very unsatisfactory in vivo host for immunological studies, is even less suitable for studying the HIV. Add to these factors the latency exhibited by HIV in inducing AIDS symptoms, the low proportion of lymphocytes even in fully infected AIDS patients which contain HIV (1:10,000 to 1:100,000), the possibility that HIV induces an autoimmune response, and the probable induction by HIV of an autoantibody which reacts with surface protein on HIV-infected T4 cells, suppressing the proliferation of T4 cells and inducing cytotoxicity of T4 cells, and it becomes apparent that the human body is not a suitable subject for most immunological studies on HIV. In addition, testing of trial drugs on AIDS patients or HIV vaccines on healthy patients carries risk factors which may make such studies unethical in man.
There has, of course, been an enormous amount of work done on the immunology of HIV and on methods for detecting the virus. For example, a diagnostic method for AIDS and pre-AIDS, and a kit therefor involving the formation of an antigen-antibody complex has been described by Axler-Blin, et al., U.S. Pat. No. 4,708,818. Virus are isolated from the sera of patients afflicted with lymphadenopathy syndrome (LAS) or AIDS. Immunodeficiency virus (HIV), viral extract, structural proteins and other fractions of the retrovirus are recognized by the sera of such patients.
The human immune system responds directly or indirectly the introduction of substances foreign to the human body into the human body by injection, ingestion or otherwise. Some such responses are not immunological, in the strictest sense, but the responses which appear in the immune system may be used as indicative of the efficacy or toxicity of the foreign substance. In the evaluation of new drugs it has been necessary to test the drug in three, or sometimes more, species, including in many cases in primates, before the drug could be considered for testing on humans. One of the features of this invention is that a relatively inexpensive test animal is made available for testing the response of the human immune system to substances foreign to the human without having to subject humans to such testing.
In the exemplary embodiment of the present invention, the cellular components of the human immune system are injected into a mutant strain of mice; these mice have severe combined immunodeficiency disease (SCID), are well-known and widely used laboratory animals, and have been, used in many immunological and oncological studies, see, for example, Dorshkind K., et al., "Functional Status of Cells from Lymphold and Myeloid Tissues in Mice with Severe Combined Immunodeficiency Disease," J. Immunol., 132(4):1804-1808, 1984; Czitrom,A. A., et al., "The Function of Antigen-Presenting Cells in Mice with Severe Combined Immunodeficiency," Immunol., 134(4):2276-2280, 1985; Dorshkind, K. et al., "Natural Killer Cells are Present in Mice with Severe Combined Immunodeficiency," J. Immunol., 134(6):3798-3801, 1;985; Custer, R. P., "Severe Combined Immunodeficiency in the Mouse Pathology Reconstitution Neoplasms," Am. J. Pathol., 120(3):464-477, 1985; Ware, C. F., "Human Rat or Mouse Hybridomas Secret High Levels of Monoclonal Antibodies Following Transplantation into Mice with Severe Combined Immunodeficiency Disease," J. Immunol. Methods, 85(2):353-362, 1985. The latter publication reports the investigation of mice with severe combined immunodeficiency [disease (SCID) for their ability to grow xenogeneic hybridomas of mouse, rat and human origin. Two rat x mouse hybridoma lines (187.1.10 and 3B9) and one mouse x mouse hybridoma (2D9) grown (as ascites tumors) in pristane-treated SCID mice as ascites tumors showed a 100-200-fold increase in monoclonal antibody levels Over the amount produced in vitro with a total yield up to 0.5 g of antibody per animal. A human x human hybridoma, CLL-11-D1, exhibited a 1000-fold increase in human immunoglobuin levels in ascites (1.3 mg/ml) as compared to that obtained in tissue culture.
Mice with severe combined immunodeficiency syndrome (SCID) exhibit an impairment in both T and B cell maturation, Lauzon, R. J., et al., "An Expanded Population of Natural Killer Cells in Mice with Severe Combined Immunodeficiency Lack Rearrangement and Expression of T Cell Receptor Genes," J. Exp. Med., 164(5): 1797-1802, 1986; Dorshkind, K. et al., "Lymphold Bone Marrow Cultures can Reconstitute Heterogenous B and T Cell-Dependent Responses in Severe Combined Immunodeficient Mice," J. Immunol., 137(11):3457-3463, 1986.
Other laboratory animals which possess little or no immune system of their own, or which have been treated with drugs or radiation, or produced through traditional genetic development or genetic engineering to have either a suppressed immune system, a weakened immune system or a modified immune system, or no immune system at all, such as, e.g. SCID horses and other SCID animals and potentially even AIDS infected animals in which AIDS has been arrested after destruction or inactivation of the animals' immune system may be considered as laboratory animal candidates for use in the present invention and thus functionally the same as or equivalent to SCID animals. A number of such animals are known, e.g. SCID mice, SCID horses, nude mice, etc., and equivalent animal candidates will undoubtedly come available as needed. SCID horses, for example, have been widely studied, see e.g. Perryman L. E., McGuire, T. C., Torbeck, R. L., and Magnuson, N. S., "Evaluation of Fetal Liver Cell Transplantation for Immuno-Reconstitution," Clin. Immunol. Immunopath., 23(1):1-9, 1982. Perryman, et al., ibid., report, for example, on efforts to transfer the immune system from a normal horse to an SCID horse in which fourteen foals with severe combined immunodeficiency (SCID) were given single-cell suspensions prepared from livers of equine fetuses 45-201 days gestational age. None of the foals demonstrated functional engraftment in terms of identifiable donor lymphocytes, increased peripheral blood lymphocyte counts, development of phytolectin- or antigen-responsive lymphocytes, or the ability to synthesize Ig and specific antibodies. Such a transfer of at least part of the immunological system was, previously, apparently transferred within the same species using this technique employing fetal liver and thymus cells in combination. No transfer of the immunological system of one species to another species is known to have been reported.
Another need for a model where the cellular components of a human immune system can be made to function in mice, or other laboratory animals, is for studies of tumors and ontogenesis involving the human immune system. Malignancies can be induced in laboratory animals by the implantation of malignant cells or by exposing the animal to carcinogens. In the first instance, the type of malignant cell implanted defines the malignancy and in the second instance the nature of the malignancy to be expected is principally a function of the point of exposure of the animal to the carcinogen. For example, SCID mice have been used to propagate a human lung tumor, Reddy, S., et al., "Human Lung Tumor Growth established in the Lung and Subcutaneous Tissue of Mice with Severe Combined Immunodeficiency," Cancer Res., 47(9):2456-2460, 1987. Thus, a feature of the present invention is that under certain conditions, malignancy of human lymphocytes can be induced within a relatively short period of time, within about eight weeks, thus providing a rapid and attractive model for the study of malignant transformation of human lymphocytes.